The Screening And Functional Verification Of Differentially Expressed Genes In Dermal Papilla Cells Of Different Lambskin Patterns Of Hu Sheep

Hu sheep is a rare sheep breed in the world with white lambskin.Its lambskin is popular among customers for the unique wave like pattern.However,the quality of Hu sheep lambskin decreased gradually due to the unplanned hybridization.Hence,it’s urgent to protect the germplasm resources of its lambskin effectively,and researching the molecular mechanism of the lambskin pattern formation is vital to the protection and utilization of the species.According to the width of the lambskin pattern and the curvature of the wool fiber,Hu sheep lambskin can be divided into four types:small flower(or extremely curved)type,medium flower(or medium curved)type,large flower(or little curved)type and straight type.Among the types above,the small flower type has the best quality and the straight type is the worst.Previously,there have been transcriptome sequencing studies on different Hu sheep lambskin patterns,but the results of traditional transcriptome sequencing were the average of all cells in the whole hair follicle,so it is impossible to analyze the expression of dermal papilla cells(DPCs),which play a key role in hair growth and hair follicle cycle regulation.But with the development of single cell RNA sequencing(scRNA-seq),this problem is being solved.In this study,the skin of small flower type and straight type was sequenced and analysed using scRNA-seq.Then we selected PAPPA2,one of the 274 differentially expressed genes in DPCs of the two types above,to carry out in-depth functional research.We also primarily clarified the function of PAPPA2 in the formation of different patterns and provide a new direction for further research on the mechanism of the formation of Hu sheep lambskin patterns.The major findings are as follows:(1)We successfully completed the analysis and validation of scRNA-seq in Hu sheep lambskin tissue,and screened the differentially expressed genes of DPCs in different patterns.In order to screen the differentially expressed genes of specific cells from Hu sheep skin with different patterns,the scRNA-seq,provided by 10x genomic Co.,was used to detect the transcriptional expression level of 8567 skin cells in small flower group and 7263 skin cells in straight group.Then,after the completion of UAMP cluster analysis,the marker genes of 19 cell clusters were detailed analysed to confirm the exact types of the cells.Finally,the main cell groups of hair follicles were stained with CUX1、KRT71、PCNA、VDR、VC AN、α-SMA and VIM by immunofluorescence to verify the results of cell grouping.In the above experiments,the sequenced cells were successfully classified into two possible novel cells and other 14 types known cells,including DPCs,matrix cells(MX),inner root sheath cells(IRS)and outer root sheath cells(ORS).Several novel possible marker genes of sheep skin cells were proposed through data analysis.At last,we analyzed the gene differential expression of DPCs from small flower and straight hair,and 274 extremely significant differentially expressed genes(P<0.01)were found from these two types of DPCs,and finally PAPPA2 was selected for research.(2)We successfully isolated and cultured DPCs from Hu sheep in vitro by using an improved method.In order to optimize the isolation and culture method of DPCs from Hu sheep lambs in vitro and provide research materials for subsequent experiments,this study optimized the isolation method of DPCs based on the characteristics of Hu sheep lambskin tissue.We reduced the demand of manpower and time by elimating steps like dispase digestion and directly culturing the dermal papilla tissues.After cell purification,we confirm the types of isolated cells through morphological identification and immunofluorescence identification.The results of morphological identification found that the cells isolated in this study had agglutination characteristics consistent with DPCs,and the immunofluorescence results of VIM,α-SMA,IGFBP3 and VCAN proteins were positive.These two results indicates that the isolated cells are DPCs.In addition,when DPCs were cultured in vitro,we found that IGFBP3 and VCAN had similar local high expression phenomenon which indicates that IGFBP3 and VCAN participate in the regulation of DPCs aggregation in vitro.(3)PAPPA2 regulates IGFBP5 in Hu sheep DPCs and enhances DPCs proliferation.In order to explore the potential molecular mechanism of DPCs influencing the skin pattern type of Hu sheep,this study selected PAPPA2 as the follow-up research focus by consulting the literature and combining with the results of differentially expressed genes of DPCs in scRNA-seq.First of all,RT-qPCR was used to verify the differential expression of PAPPA2 in DPCs between small flower type and straight type(P<0.01).Then,PAPPA2 and IGFBP5 eukaryotic overexpression vectors(pPAPPA2-O and pIGFBP5-O)were constructed and PAPPA2 small interference RNA(siRNA-1/2/3)was designed and synthesized.After transfecting pPAPPA2-O,pIGFBP5-O and siRNA-1/2/3 in DPCs,RT-qPCR,EdU and cell cycle detection were performed to explore the effects of PAPPA2 and IGFBP5 on the proliferation of DPCs.Results of RT-qPCR showed that overexpression of PAPPA2 in DPCs could extremely significantly increase the expression of PAPPA2 and IGFBP5 at the same time(P<0.01).But overexpression of IGFBP5 only increased the expression of IGFBP5.Results of the cell cycle detection and EdU found that solely overexpressing PAPPA2 can significantly increase the proliferation ability of DPCs(P<0.05),while interfering PAPPA2 or overexpressing IGFBP5 can significantly reduce the proliferation ability of DPCs(P<0.05).Furthermore,compared with solely overexpressing IGFBP5,overexpressing both genes can extremely significantly increase the proliferation ability of DPCs(P<0.01).The above results indicate that PAPPA2 can regulate the proliferation of DPCs by regulating IGFBP5.In conclusion,firstly,this study had completed the scRNA-seq and analysis of Hu sheep skin,and 274 differentially expressed genes were screened in two patterns’ DPCs.Then,this study improved the former isolation and culture method of Hu sheep DPCs in vitro,and identified these cells with the marker genes of DPCs.It was found that IGFBP3 participate in the regulation of DPCs aggregation in vitro.At last,studies on the differentially expressed PAPPA2 revealed that it could promote the proliferation of DPCs by regulating IGFBP5,which in turn affected the formation of lambskin pattern in Hu sheep.